Beneficial Effect of a Fermented Wheat Germ Extract in Intestinal Epithelial Cells in case of Lipopolysaccharide-Evoked Inflammation.

In this study, the protective effect of a fermented wheat germ extract (FWGE) against LPS-induced inflammation and oxidative stress in IPEC-J2 porcine intestinal epithelial cells was studied. Enterocytes were treated with LPS derived from Salmonella enterica ser. Typhimurium and Escherichia coli O55:B5, O111:B4, and O127:B8 strains. Intracellular ROS level and extracellular H2O2 level were followed up by two fluorescent assays (DCFH-DA and Amplex Red). The effect of FWGE on the intestinal barrier integrity was determined by transepithelial electric resistance measurements and using a FD4 fluorescent tracer dye. IL-6 concentration of supernatants was also measured by the ELISA method. Our data revealed that FWGE had a significant lowering effect on the inflammatory response especially related to oxidative stress. Treatment with FWGE (1-2%) significantly decreased the level of intracellular ROS compared to LPS-treated cells. Furthermore, LPS-triggered partial disruption of epithelial integrity was reduced after FWGE application.

Effects of Lactobacillus plantarum 2142 and sodium n-butyrate in lipopolysaccharide-triggered inflammation: comparison of a porcine intestinal epithelial cell line and primary hepatocyte monocultures with a porcine enterohepatic co-culture system.

This study was based on our previously developed double-layered enterohepatic co-culture system, composed of nontumorigenic porcine intestinal epithelial cell line (IPEC-J2) and primary culture of porcine hepatocytes. The anti-inflammatory effect of spent culture supernatant of Lactobacillus plantarum 2142 (Lp2142; 13.3%) and sodium n-butyrate (2 mM) was tested on IPEC-J2 and hepatocyte monocultures as well as on the gut-liver co-culture. To mimic inflammation, lipopolysaccharide (LPS; 1 and 10 µg/mL) was applied. Production of IL-8 and IL-6 was measured as a marker of inflammatory responses. The paracellular permeability of the intestinal epithelium was also monitored by fluoresceinisothiocyanate-labeled dextran 4 assay. Significant increase of IL-8 concentration was observed in the IPEC-J2 monoculture (P < 0.01) while the level of IL-6 was not changed following LPS treatment. Concentration of IL-8 and IL-6 was grown significantly in hepatocyte monocultures (P < 0.05 and P < 0.001) as well as in the co-culture after 10 µg/mL LPS treatment (P < 0.001 and P < 0.001). One microgram per milliliter LPS caused elevated IL-8 level in the co-culture (P < 0.001) and in the hepatocyte monoculture (P < 0.01), while it caused increased IL-6 level only in the hepatocytes (P < 0.001). Production of IL-8 was significantly decreased by butyrate in case of 1 µg/mL as well as 10 µg/mL LPS exposure in the co-culture (P < 0.001). Application of butyrate also reduced IL-6 level in the co-culture after 10 µg/mL LPS treatment (P < 0.01). Lactobacillus plantarum 2142 decreased IL-8 level after incubation with 1 µg/mL LPS (P < 0.001), while in case of 10 µg/mL LPS treatment only a marginal lowering in IL-8 (P = 0.064) release was measured. The IL-6 concentration was significantly reduced (P < 0.01 in case of 1 µg/mL LPS treatment) by Lp2142 in the co-culture. Contrarily, the elevated IL-8 and IL-6 level of hepatocytes has not been reduced in case of either butyrate or Lp2142 addition. The enterohepatic co-culture model offers a possibility for fast and reliable screening of new candidates against enteric inflammation, which are of special interest in porcine medicine and health management. According to our results, Lp2142 and butyrate both seem to be effective as anti-inflammatory agents in LPS-triggered inflammatory response, tested in the gut-liver co-culture model.

Variability of microcystins and its synthetase gene cluster in Microcystis and Planktothrix waterblooms in shallow lakes of Hungary.

Waterbloom samples of Microcystis aeruginosa and Planktothrix agardhii were collected from a variety of ponds, lakes and reservoirs in Hungary. Samples were tested with matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry (MALDI-TOF MS) to identify the microcystin forms. The concentration of the microcystins was measured with capillary electrophoresis and the toxicity was tested by sinapis test. DNA was extracted from the samples and tested using a range of primers linked to the biosynthesis of microcystin. All of the fourteen collected samples gave positive results for the presence of the mcy genes with PCR products with sizes between of 425 and 955 bp, respectively, indicating the presence of the genes implicated in the production of microcystins. The results showed that a wide range of microcystin (MC) forms were detected in the Microcystis containing samples, among which MC-LR, -RR, and -YR were the most common. The highest MC concentration was 15,701 mg g-1, which was detected in an angling pond. The samples containing Planktothrix agardhii were less toxic, and the most common form in this species was the Asp3-MC-LR.

Lactobacillus plantarum 2142 prevents intestinal oxidative stress in optimized in vitro systems.

Recently, there has been a growing interest to replace antibiotics' administration with the application of probiotics. The aim of our investigations was to reveal the influence of spent culture supernatant of Lactobacillus plantarum 2142 on the response of enterocytes to oxidative stress, and the spent culture supernatant's ability to protect them from oxidative injury. The experiments were performed on non-carcinogenic porcine epithelial cell line, IPEC-J2 isolated from a neonatal piglet and on human colon adenocarcinoma cell line, Caco-2. The cells cultured on membrane inserts were treated with millimolar hydrogen peroxide solution to provoke oxidative stress. The peroxide-triggered cell response profile was evaluated via determination of change in transepithelial electrical resistance, quantification of extent of cell death by 4',6-diamidino-2 phenylindole (DAPI) staining and via estimation of proinflammatory cytokine, IL-8 production using ELISA technique. Non-starter lactobacilli supernatant-mediated inhibition of peroxide-triggered upregulation of IL-8 production confirmed the antiinflammatory properties of active metabolites produced by Lactobacillus plantarum 2142 in acute oxidative stress.

Kinetics of the cellular immune response following closed head injury.

BACKGROUND: The contribution of brain edema to brain swelling in cases of traumatic brain injury (TBI) remains a critical problem. We believe that inflammatory reactions may play a fundamental role in brain swelling following a head injury. Although possible roles of microglia activation and the release of mediators have been suggested, direct evidence of cellular immune reactivity in diffuse brain injury following closed head trauma is lacking. Accordingly, the objective of this study was to assess the temporal pattern of microglia activation and lymphocyte migration in an experimental model of TBI. METHOD: An impact acceleration TBI model was utilized to induce diffuse brain damage in adult Wistar rats. The animals were separated into three groups: unoperated controls, sham-operated controls and trauma group. At various times after TBI induction (5 min-24 h), rats were perfused transcardially. Sagittal brain sections were analyzed with immunohistochemical markers of CD3 to reveal the presence of T-lymphocytes, and by immunochemistry for the detection of CD11b to reveal microglia activation within the brain parenchyma. FINDINGS: In the control groups, scattered T-cells were found in the brain parenchyma. In the trauma group, TBI induced microglia activation and a transient biphasic T-cell infiltration of the brain parenchyma in all regions was found, beginning as early as 30 min post injury and reaching its maximum values at 45 min and 3 h after trauma induction. CONCLUSION: These results lead us to suggest that the acute response to severe head trauma with early edema formation is likely to be associated with inflammatory events which might be triggered by activated microglia and infiltrating lymphocytes. It is difficult to overestimate the clinical significance of these observations, as the early and targeted treatment of patients with severe head injuries with immunosuppressive medication may result in a far more favorable outcome.

Spectrin breakdown products in the cerebrospinal fluid in severe head injury--preliminary observations.

BACKGROUND: Calcium-induced proteolytic processes are considered key players in the progressive pathobiology of traumatic brain injury (TBI). Activation of calpain and caspases after TBI leads to the cleavage of cytoskeletal proteins such as non-erythroid alpha II-spectrin. Recent reports demonstrate that the levels of spectrin and spectrin breakdown products (SBDPs) are elevated in vitro after mechanical injury, in the cerebrospinal fluid (CSF) and brain tissue following experimental TBI, and in human brain tissue after TBI. METHODS: This study was initiated to detect spectrin and SBDP accumulation in the ventricular CSF of 12 severe TBI-patients with raised intracranial pressure (ICP). Nine patients with non-traumatically elevated ICP and 5 undergoing diagnostic lumbar puncture (LP) served as controls. Intact spectrin and calpain and caspase specific SBDPs in CSF collected once a day over a several day period were assessed via Western blot analysis. Parameters of severity and outcome such as ICP, Glasgow Coma Scale and Glasgow Outcome Scale were also monitored in order to reveal a potential correlation between these CSF markers and clinical parameters. RESULTS: In control patients undergone LP no immunoreactivity was detected. Non-erythroid alpha-II-spectrin and SBDP occurred more frequently and their level was significantly higher in the CSF of TBI patients than in other pathological conditions associated with raised ICP. Those TBI patients followed for several days post-injury revealed a consistent temporal pattern for protein accumulation with the highest level achieved on the 2(nd) -3(rd) days after TBI. CONCLUSION: Elevation of calpain and caspase specific SBDPs is a significant finding in TBI patients indicating that intact brain spectrin- and SBDP-levels are closely associated with the specific neurochemical processes evoked by TBI. The results strongly support the potential utility of these surrogate markers in the clinical monitoring of patients with severe TBI and provide further evidence of the role of calcium-induced, calpain- and caspase-mediated structural proteolysis in TBI.

Preinjury administration of the calpain inhibitor MDL-28170 attenuates traumatically induced axonal injury.

Traumatic brain injury (TBI) evokes diffuse (traumatic) axonal injury (TAI), which contributes to morbidity and mortality. Damaged axons display progressive alterations gradually evolving to axonal disconnection. In severe TAI, the tensile forces of injury lead to a focal influx of Ca2+, initiating a series of proteolytic processes wherein the cysteine proteases, calpain and caspase modify the axonal cytoskeleton, causing irreversible damage over time postinjury. Although several studies have demonstrated that the systemic administration of calpain inhibitors reduces the extent of ischemic and traumatic contusional injury a direct beneficial effect on TAI has not been established to date. The current study was initiated to address this issue in an impact acceleration rat-TBI model in order to provide further evidence on the contribution of calpain-mediated proteolytic processes in the pathogenesis of TAI, while further supporting the utility of calpain-inhibitors. A single tail vein bolus injection of 30 mg/kg MDL-28170 was administered to Wistar rats 30 min preinjury. After injury the rats were allowed to survive 120 min when they were perfused with aldehydes. Brains were processed for immunohistochemical localization of damaged axonal profiles displaying either amyloid precursor protein (APP)- or RMO-14-immunoreactivity (IR), both considered markers of specific features of TAI. Digital data acquisition and statistical analysis demonstrated that preinjury administration of MDL-28170 significantly reduced the mean number of damaged RMO-14- as well as APP-IR axonal profiles in the brainstem fiber tracts analyzed. These results further underscore the role of calpain-mediated proteolytic processes in the pathogenesis of DAI and support the potential use of cell permeable calpain-inhibitors as a rational therapeutic approach in TBI.

Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance.

It was earlier established that one of the primary morphopathological consequences of experimental traumatic brain injury is a dramatic reduction in the distances between the neurofilaments (cytoskeletal compaction) inside a number of axon segments that appear to be randomly distributed among normal axons in an otherwise undamaged parenchymal environment. The present results demonstrate that the cytoskeletal compaction instantly induces argyrophilia, thereby rendering possible selective visualisation of the affected axon segments for light microscopy through use of a special silver staining method. On combination of this method with electron microscopy, it was revealed that the cytoskeletal compaction is completed in much shorter times and extends to much longer axon segments than previously assumed.

Association between gastric cancer mortality and nitrate content of drinking water: ecological study on small area inequalities.

The carcinogenic feature of N-nitroso compounds has been well established. Similarly, the transformation of ingested nitrate to N-nitroso compounds in the stomach has been thoroughly documented, nevertheless nitrates' carcinogenic effect has not been proved convincingly in human. The present study was aimed to investigate a population of small villages provided by drinking water with high and widely variable nitrate content (72 mg/l median, 290.7 mg/l 95-percentile concentration). Empirical Bayes estimates for settlement-specific age-, sex-, and year-standardised mortality ratios of gastric cancer (GC) were related to the settlement level average nitrate concentrations in drinking water controlling for confounding effects of smoking, ethnicity and education. The log-transformed average nitrate concentration showed significant positive association with stomach cancer mortality in linear regression analysis (p = 0.014). The settlements were aggregated according to the nitrate concentration into 10-percentile groups and the standardised mortality ratios (SMRs) were calculated. Those groups with higher than 88 mg/l average nitrate concentration showed substantial risk elevation and the log-transformed exposure variables proved to be significant predictors of mortality (p = 0.032) at this level of aggregation also. The association seemed to be fairly strong (r2 = 0.46). Although this investigation constituting an ecological study has certain limitations, it supports the hypothesis that the high level of nitrate in drinking water is involved in the development of GC.

Health status and risk factors of seminomadic pastoralists in Mongolia: a geographical approach.

The particular lifestyle of nomadic or seminomadic people has much to do with their health status. This discussion of the conceptual basis and some preliminary results of the 1992-94 health status and risk factor survey in Mongolia serves to highlight some of the relationships existing between the general health status and potential risk factors observed among pastoral nomads. In addition to graphic description of the data, a statistical analysis suggests significant associations between certain health status indicators and gender, location, lifestyle factors (e.g. smoking) socio-economic status, preventive health care and the physical environment. With regard to locational factors, there are strong regional differences in a wide-ranging number of health status indicators. The results of this study, obtained as they were at the threshold of Mongolia's economic and political transformation, will serve as a baseline against which to evaluate future changes in the health of Mongolians.

Molecular modelling of xylose isomerase catalysis: the role of electrostatics and charge transfer to metals.

The two main steps of the mechanism of xylose-xylulose conversion catalysed by D-xylose isomerase, the ring opening of xylose and the isomerization of the opened product by hydride transfer, were investigated by molecular mechanical and molecular orbital techniques. The activation energies calculated for these reactions clearly showed that hydrogen transfer is the rate-determining step of the enzymatic isomerization and that Mg2+ ions activate whereas Zn2+ ions inhibit the reaction, in agreement with the experiments. The remarkable differences between the net charges of these ions found by molecular orbital calculations and the inspection of the protein electrostatic potential around the reaction intermediates indicate that the main role of bivalent metal ions should be the electrostatic stabilization of the substrate transition states. In order to propose a more detailed mechanism, an attempt was made to clarify the effects of nearby residues (e.g. His54, Asp57, Lys183, Asp257) in the reaction. Different isomerization mechanisms, such as through an enediol intermediate, were examined and could be excluded, in addition to the charge-relay mechanism during the ring opening.